Anti-cancer compounds

ABSTRACT

Compounds of formula (I) ##STR1## in which R 1 , R 2 , R 3  and R 4  are each separately selected from hydrogen, X, NH--A--NHR and NH--A--N(O)R&#39;R&#34; wherein X is hydroxy, halogeno, amino, C 1-4  alkoxy or C 2-8  alkanoyloxy, A is a C 2-4  alkylene group with a chain length between NH and NHR or N(O)R&#39;R&#34; of at least 2 carbon atoms and R, R&#39; and R&#34; are each separately selected from C 1-4  alkyl groups and C 2-4  hydroxyalkyl and C 2-4  dihydroxyalkyl groups in which the carbon atom attached to the nitrogen atom does not carry a hydroxy group and no carbon atom is substituted by two hydroxy groups, or R&#39; and R&#34; together are a C 2-6  alkylene group which with the nitrogen atom to which R&#34; and R&#34; are attached forms a heterocyclic group having 3 to 7 atoms in the ring, but with the proviso that at least one of R 1  to R 4  is a group NH--A--N(O)R&#39;R&#34;, and physiologically acceptable salts thereof are of value in the treatment of cancer.

This invention relates to novel anthraquinones which are of particularvalue in the treatment of cancer.

A wide variety of aminoalkylamino anthraquinones(aminoalkylaminoanthracene-9,10-diones) has been described for use aschemotherapeutic agents for the treatment of cancer, perhaps the mostactive being the compound mitoxantrone (mitozantrone) of formula##STR2## which is the subject of U.S. Pat. No. 4,197,249 and U.K. Patent2,004,293B. However, in common with other cytotoxic chemotherapeuticagents the aminoalkylamino anthraquinones have the disadvantgage thattheir activity is not confined to neoplastic cells and they thereforeexhibit various undesirable side effects including, to a greater orlesser extent among the different compounds, myelosuppression andcardiotoxicity.

It is an object of the present invention to provide a group ofanthraquinone pro-drugs which are of lesser cytotoxicity than the drugitself, preferably being substantially non-cytotoxic, the pro-drugsbeing converted in vivo under the anaerobic conditions within neoplastictissue to the cytotoxic drug thereby mitigating the side effects ofadministering that drug directly.

Accordingly the present invention comprises a compound of formula (I)##STR3## in which R₁, R₂, R₃ and R₄ are each separately selected fromhydrogen, X, NH--A--NHR and NH--A--N(O)R'R" wherein X is hydroxy,halogeno amino, C₁₋₄ alkoxy or C₂₋₈ alkanoyloxy, A is a C₂₋₄ alkylenegroup with a chain length between NH and NHR or N(O)R'R" of at least 2carbon atoms and R, R' and R" are each separately selected from C₁₋₄alkyl groups and C₂₋₄ hydroxyalkyl and C₂₋₄ dihydroxyalkyl groups inwhich the carbon atom attached to the nitrogen atom does not carry ahydroxy group and no carbon atom is substituted by two hydroxy groups,or R' and R" together are a C₂₋₆ alkylene group which with the nitrogenatom to which R' and R" are attached forms a heterocyclic group having 3to 7 atoms in the ring, but with the proviso that at least one of R₁ toR₄ is a group NH--A--N(O)R'R", the compound optionally being in the formof a physiologically acceptable salt.

The compounds of formula (I) contain at least one substituent groupNH--A--N(O) R'R" having the terminal, tertiary nitrogen atom in N-oxideform. Although groups of this type are not unknown, for example beingdescribed in European Patent Application A-0 145 226 as one of thevarious alternative forms of substituent in a group of substitutednitroacridones, it had not previously been appreciated that such asubstituent confers valuable properties as compared with the compoundcontaining the corresponding group NH--A--NR'R" in which the terminal,tertiary nitrogen atom is not in N-oxide form. Thus, such N-oxides arebioreductively activated within neoplastic tissue to form the cytotoxiccompound containing an NH--A--NR'R" group thereby providing the desiredanti-cancer activity of this compound but with mitigation of itsundesired side effects.

It will be seen that in addition to the one or more substituentsNH--A--N(O)R'R" in N-oxide form the compounds (I) may contain one ormore substituents NH--A--NHR. Whilst these compounds, as compared withthose containing no group NH--A--NHR, may exhibit some degree ofcytotoxicity and are thus less preferred, this will nonetheless be at alower level than the corresponding compound in which none of theaminoalkylamino groups is in N-oxide form and full cytotoxicity willonly be expressed on conversion of the group(s) NH--A--N(O)R'R" togroup(s) NH--A--NR'R".

As regards the groups NH--A--NHR and NH--A--N(O)R'R", A may be branchedbut is conveniently a straight chain alkylene group, i.e.tetramethylene, especially trimethylene, or particularly ethylene.

R, R' and R" may also have a branched carbon chain but are convenientlystraight chain whether they are alkyl groups or hydroxy-substitutedalkyl groups. When R, R' or R" is a monohydroxyalkyl group this isconveniently substituted terminally and when R, R' or R" is adihydroxyalkyl group this is conveniently substituted terminally by oneof the hydroxy groups. When R, R' and R" are alkyl the preference is fora group of three or especially two or one carbon atoms and when R, R'and R" are hydroxy-substituted alkyl the preference is for the alkylgroup to be of three carbon atoms or, in the case of a monohydroxyalkylgroup, alternatively of two carbon atoms. Examples of preferredindividual groups R, R' and R" are CH₃, CH₂ CH₂ CH₃, CH₂ CH₂ OH, CH₂ CH₂CH₂ OH, CH(CH₃)CH₂ OH, and CH₂ CHOHCH₂ OH and especially CH₂ CH₃. WhilstR' and R" will more usually be identical there can be certain advantagesas described hereinafter in having non-identical groups R' and R".

Alternatively, as indicated, R' and R" together with the nitrogen atomto which they are attached may represent a heterocyclic group--N(CH₂)_(n) where n is 2 to 6, i.e. aziridin-1-yl, azetidin-1-yl,pyrrolidin-1-yl, piperidin-1-yl and perhydroazepin-1-yl, the smallergroups such as azetidin-1-yl and especially aziridin-1-yl being of mostinterest.

Specific groups NH--A--NHR of particular interest are NH--(CH₂)₂--NHCH₃, NH--(CH₂)₂ --NHCH₂ CH₂ CH₂ OH, NH--(CH₂)₂ --NHCH(CH₃)CH₂ OH,NH--(CH₂)₂ --NHCH₂ CHOHCH₂ OH, especially NH--(CH₂)₂ --NHCH₂ CH₂ OH andparticularly NH--(CH₂)₂ --NHC₂ H₅, whilst specific groupsNH--A--N(O)R'R" of particular interest are NH--(CH₂)₂ --N(O)(CH₃)C₂ H₅,NH--(CH₂)₂ --N(O)(CH₂ CH₂ OH)₂, NH--(CH₂)₂ --N(O)(CH₂ CH₂ CH₂ OH)₂,NH--(CH₂)₂ --N(O)(CH(CH₃)CH₂ OH)₂, NH--(CH₂)₂ --N(O)(CH₂ CHOHCH₂ OH)₂,especially NH--(CH₂)₂ --N(O)(CH₃)₂ and particularly NH--(CH₂)₂ --N(O)(C₂H₅)₂.

As regards the groups X, the halogeno groups are preferably bromo andespecially chloro. Alkoxy and alkanoyloxy groups X may be branched orespecially straight chain and are conveniently of 1 or 2 carbon atomsfor the alkyl groups and of 2 or 3 carbon atoms for the alkanoyl groups.Examples of such groups X are therefore chloro, amino and especiallymethoxy, ethoxy, acetyl and propionyl. However hydroxy groups arepreferred as the group or groups X.

Formula (II) illustrates the system used for numbering the variouspositions of the anthracene-9,10-dione ring system. ##STR4##

It will be seen from this formula that, due to the symmetrical nature ofthe molecule, certain substitution patterns are equivalent, for example1,4 and 5,8. Preferences as to positions of substitution are expressedherein by identifying groups R₁ to R₄ which are other than hydrogen inthe order R₁, R₂, R₃, R₄ and, as is conventional practice, byidentifying substituted positions in the order 1. 2, 3, 4, 5, 6, 7, 8.Thus, for example, the compound having one substituent only at a ringposition meta to an oxo group is identified as having that substituentas a group R₁ at position 1 and groups R₂, R₃ and R₄ which are hydrogen.

Any of positions 1, 2, 3, 4, 5, 6, 7 and 8 in the compound (I) may besubstituted by a group NH--A--N(O)R'R" but positions 1, 4, 5 and 8 areof most interest for substitution by such a group and, indeed, also bythe other substituent groups. At least one group NH--A--N(O)R'R" in thecompound (I) is conveniently at a position meta to an oxo group (so thatR₁ is a group NH--A--N(O)R'R" at the 1-position). However, the compound(I) may contain more than one group NH--A--N(O)R'R", which may bedifferent as regards A and/or R' and R" but which are convenientlyidentical. Although four or more, particularly three, of such groups maybe present, preferred compounds contain two such groups, convenientlywith a 1,8, especially a 1,4 and particularly a 1,5 substitution patternfor these groups (so that R₁ is a group NH--A--N(O)R'R" at the1-position together with either R₂ being a group NH--A--N(O)R'R" at the4-position or R₃ being a group NH--A--N(O)R'R" at the 5- or 8-position).

The compounds (I) may contain one to three groups NH--A--NHR butconveniently no more than two and preferably no more than one of suchgroups is present. Where one or more groups NH--A--NHR are present thesemay differ or not as regards A and/or R between each other and ascompared with A and R' and R" in the group(s) NH--A--N(O)R'R" which arepresent. Preferably, however, each group NH--A--NHR is identical wheremore than one of these is present. Where only one group NH--A--N(O)R'R"is present or more than one of such groups which are the same arepresent, a possibility is for any group NH--A--NHR to be the same as thegroup(s) NH--A--N(O)R'R" as regards A and conveniently for R to be thesame as R' and/or R". Where one or more groups NH--A--NHR is presentthere is preferably one such group at a position meta to an oxo group.Compounds containing such a group are conveniently substituted at one ormore of positions 1, 4, 5 and 8 by this group or groups. Preferredcompounds of this type contain only one group NH--A--NHR and one groupNH--A--N(O)R'R", compounds of particular interest having a groupNH--A--N(O)R'R" at position 1 and a group NH--A--NHR at position 8 orespecially at position 5 and particularly at position 4, optionally withsubstitution by a group or groups X, particularly hydroxy, at one orboth of the other of these positions.

Conveniently the compounds (I) contain also at least one and preferablytwo groups X, particularly hydroxy groups. Once again, a group X may beat any of positions 1, 2, 3, 4, 5, 6, 7 and 8 but such a group mayconveniently being at one or two of the positions 1, 4, 5 and 8,providing these are not occupied by a group NH--A--NHR orNH--A--N(O)R'R". Conveniently three groups X may be present when onlyone group NH--A--N(O)R'R" and no group NH--A--NHR is present and twogroups X may be present when two groups NH--A--N(O)R'R" or one groupNH--A--N(O)R'R" and one group NH--A--NHR are present, such groups Xbeing the same or different. Preferably position 1 is occupied by agroup NH--A--N(O)R'R" and the positions 4, 5 and 8 are occupied by agroup X, particularly a hydroxy group in each case, or one of thesethree positions is occupied by another group NH--A--N(O)R'R" or a groupNH--A--NHR and the remaining two positions are occupied by a group X,particularly a hydroxy group in each case. Compounds having a group X ateach of positions 5 and 8, particularly a hydroxy group in each case,are preferred, for example those indicated under (1), (5) and (7) below.

Compounds of particular interest thus have one of:

(1) R₁ =NH--A--N(O)R'R" (position 1), R₂ =H, R₃ =R₄ =OH (positions 5 and8);

(2) R₁ =NH--A--N(O)R'R" (position 1), R₂ =OH (position 4), R₃ =OH(position 5 or position 8), R₄ =H;

(3) R₁ =NH--A--N(O)R'R" (position 1), R₂ =R₃ =R₄ =OH (positions 4, 5 and8);

(4) R₁ =R₃ =NH--A--N(O)R'R" (positions 1 and 8, conveniently beingidentical groups), R₂ =R₄ =OH (positions 4 and 5 respectively);

(5) R₁ =R₂ =NH--A--N(O)R'R" (positions 1 and 4, conveniently beingidentical groups), R₃ =R₄ =OH (positions 5 and 8); (6) R₁ =R₃=NH--A--N(O)R'R" (positions 1 and 5, conveniently being identicalgroups), R₂ =R₄ =OH (positions 4 and 8);

(7) R₁ =NH--A--N(O) R'R" at position 1, R₂ =NH--A--NHR at position 4, R₃=R₄ =OH at positions 5 and 8;

(8) R₁ =NH--A--N(O)R'R" at position 1, R₂ =OH at position 4, R₃=NH--A--NHR at position 5 and R₄ =OH at position 8, or

(9) R₁ =NH--A--N(O)R'R" at position 1, R₂ =R₃ =OH at positions 4 and 5,and R₄ =NH--A--HR at position 8. Of these, the compounds of types (1) to(6), particularly of type (5) and especially of type (6) are preferred.

Specific compounds (I) according to the present invention include thosecompounds of types (1) to (9) just listed in which the or each groupNH--A--N(O)R'R" and any group NH--A--NHR has A=(CH₂)₂ and R, R' and R"each separately=CH₃, CH₂ CH₂ CH₃, CH₂ CH₂ OH, CH₂ CH₂ CH₂ OH, CH(CH₃)CH₂OH or CH₂ CHOHCH₂ OH or particularly CH₂ CH₃. Preferably R' and R" areidentical for each group NH--A--N(O)R'R" and conveniently where twogroups NH--A--N(O)R'R" are present these are identical. Particularlypreferred specific compounds are those of formula (III) and particularlyof formula (IV) and their analogues in which the two methyl groups inN(O)(CH₃)₂ are replaced by two n-propyl, 2-hydroxyethyl,2-hydroxypropyl, 3-hydroxypropyl, 2,3-dihydroxypropyl or particularlyethyl groups, such compounds being in the free base or a salt form.

A further group of specific compounds, which are also of some interest,consists of compounds analogous to compounds (III) and (IV) and theanalogues thereof just mentioned but in which the NH--(CH₂)₂ --N(O)R'R"group at position 4 or 5 is replaced by a group NH--(CH₂)₂ --NHCH₂ CH₂OH or a variant of such a group in which the 2-hydroxyethyl groupthereof is replaced by a methyl, n-propyl, 2hydroxypropyl,3-hydroxypropyl, 2,3-dihydroxypropyl or particularly an ethyl group.##STR5##

Certain substituents in the compounds (I) may contain one or moreasymmetric carbon atoms and the compounds will then exist instereoisomeric forms. Moreover, in the case where R' and R" aredifferent this will introduce a centre of asymmetry at the nitrogen atomin N-oxide form. It will be appreciated that one stereoisomeric form ofa compound may be of particular interest by virtue of advantageousphysical properties, for example greater solubility, or biologicalactivity.

As indicated the compounds (I) may be used in the form of aphysiologically acceptable salt which will be an acid addition salt withan organic or inorganic acid, for example with one of the acidssulphuric, phosphoric, hydrochloric, hydrobromic, sulphamic, citric,lactic, malic, succinic, tartaric, acetic, benzoic, gluconic andascorbic. Although the salts will usually have similar physiologicalproperties to the free base they may have the advantage of enhancedsolubility, etc.

The compounds (I) may conveniently be prepared through the oxidation ofthe tertiary amino group(s) of the corresponding compound in which eachgroup NH--A--N(O)R'R" in the compound (I) is in the form NH--A--NR'R".Thus, for example, anthracene-9,10-diones containing various[2-(dialkylamino)ethyl]amino, {2-[di-(hydroxyalkyl)amino]ethyl}amino and[2-(cyclic alkyleneamino)ethyl]amino groups may be oxidized to theω-N-oxides. Where appropriate the precursor compound which is oxidizedmay contain one or more modified groups X, R, R' and R" as compared withthe parent compound, the groups X, R, R' and R" corresponding to thosein the compound (I) being generated after the oxidation has beeneffected. In particular, it may be appropriate to protect the hydroxygroup(s) in groups R, R' and R" which are hydroxyalkyl or dihydroxyalkylgroups or groups X which are hydroxy during the oxidation, for exampleas an ether group such as benzyloxy, and subsequently regenerate thehydroxy group(s), for example by catalytic reduction of a benzyloxygroup. Any suitable oxidizing agent for converting a tertiary aliphaticamine to N-oxide form is suitable, for example hydrogen peroxide, oxone(potassium monopersulphate) and particularly a peracid such as3-chloroperbenzoic acid. Reaction at room temperature in the darkovernight with an excess of such an acid is usually sufficient to effectconversion to the N-oxide.

Where the compound (I) can exist in d and l forms as well as the dl forman optically active isomer may be synthesised either substantially freefrom these other forms, or at least in a major proportion by weight ascompared with them, either by using optically active reagents in thesynthesis of the compound or, particularly in the case of the opticallyactive compounds in which R' and R" are different, by resolving the dlform, especially by using an optically active inorganic or organic acidto provide two stereoisomeric salts with different physical properties.In such an instance and also where the compound (I) is used in the formof a salt the salt may be prepared by reaction of the organic base (I)with the appropriate inorganic or organic acid according to conventionalprocedures, usually by simple admixture in solution. The acid additionsalts are generally crystalline solids which are relatively soluble inwater, methanol, ethanol and similar solvents.

Accordingly the present invention comprises a process for thepreparation of a compound of formula (I) as defined hereinbefore whichcomprises oxidizing a compound of formula (Ia) ##STR6## in which R¹, R²,R³ and R⁴ correspond to R₁, R₂, R₃ and R₄, respectively, in the compound(I) but with each of the groups of the type NH--A--N(O)R'R" in thecompound (I) being instead a group NH--A--NR'R" in the compound (Ia) andone or more groups X, R, R' and R" in the compound (Ia) optionallyinstead being in a form convertible to said group or groups present inthe compound (I), and where appropriate converting the one or moremodified groups X, R, R' and R" in the compound (Ia) to the form presentin the compound (I) and/or forming an acid addition salt with aphysiologically acceptable organic or inorganic acid.

Various routes are available for the synthesis of the intermediateswhich are oxidized to the compounds (I) of the present invention. Onevery convenient procedure for the preparation of compounds having agroup NH--A--NR'R" at the 1 and 4 positions uses the appropriatelysubstituted 2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione whichis condensed with the appropriate amine R"R'N--A--NH₂, the 1,4 positionsbeing activated in the leuco compound for reaction with the amine. Sucha condensation may conveniently be effected at a temperature in a rangeof about 25° or 35° to 50° or 60° C. for one or more hours using asolvent such as methanol, ethanol, water, dimethylformamide,2-methoxyethanol, acetonitrile, nitrobenzene,N,N,N'N'-tetramethylenediamine or mixtures thereof. In some instances ahigher temperature and shorter reaction time may be appropriate, forexample with the compounds containing cyclic groups NR'R". The leucoderivative is then oxidized to the fully aromatic anthracene-9,10-dione,conveniently using air oxidation or oxidation with hydrogen peroxide,chloranil, sodium perborate or manganese dioxide.

Although leuco compounds are primarily of interest for the preparationof intermediates substituted by two NH--A--NHR'R" groups, it is possibleto use them to prepare compounds containing more than two such groups.Thus by using2,3-dihydro(leuco)-1,4,5,8-tetrahydroxyanthracene-9,10-dione and a largeexcess of an amine NH--A--NHR'R" an 8-hydroxyanthracene-9,10-dionehaving three groups NH--A--NHR'R" at the 1,4 and 5 positions may beprepared.

The leuco derivatives themselves are obtainable by heat treatment of thecorresponding fully aromatic 1,4-dihydroxyanthracene-9,10-dione,conveniently by heating at above 90° C. for 1 hour or more in a streamof nitrogen and, if necessary, in the presence of a suitable reducingagent such as sodium dithionite or zinc dust. Variousanthracene-9,10-diones, particularly hydroxyanthracene-9,10-diones, arecommercially available and various syntheses for such compounds are alsoreported in the literature. One suitable procedure for their preparationinvolves the reaction of an appropriately substituted phthalic anhydridewith hydroquinone in the presence of aluminium chloride and sodiumhydroxide at 180° C. for one hour or more. Anthracene-9,10-dionescontaining one form of substituent X can be modified to provide otherforms of substituent X so that, for example, a dione containing an aminogroup can be treated with sodium hydroxide/dithionite to yield thecorresponding hydroxy substituted compound.

Other suitable procedures for the preparation of intermediates foroxidation to the N-oxide include the reaction of the appropriate chlorosubstituted anthracene-9,10-dione with the appropriate amineR"R'N--A--NH₂, for example by heating with a excess of the amine at itsreflux temperature for one or more hours. Certain of thesechloroanthracene-9,10-diones are known and various syntheses for suchcompounds are also reported in the literature. Thus, for example,1,5-dichloro-4,8-dihydroxyanthracene-9,10-dione may be prepared byselective chlorination of 1,4,5,8-tetrahydroxyanthracene-9,10-dioneusing a stoichiometric amount of sulphuryl chloride and controlledtemperature. This precursor may then be used to prepare an intermediatehaving groups NH--A--NR'R" at the 1 and 5 positions and hydroxy groupsat the 4 and 8 positions, the hydroxy groups conveniently beingprotected during the reaction with the amine R"R'N--A--NH₂. A similarapproach is suitable for the preparation of otherchlorohydroxyanthracene-9,10-dione intermediates.

Where the compound (I) contains one or more groups NH--A--NHR inaddition to the one or more groups NH--A--NR'R" the compound mayconveniently be produced by reacting a suitable precursor as discussedabove with a mixture of amines RN--A--NH₂ and R"R'N--A--NH₂, theresultant mixture of products then being separated, for example bychromatography. Thus, for example,2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione on reaction with amixture of 2-(2-hydroxyethylamino)ethylamine and2-(diethylamino)ethylamine will yield a mixture of1,4-bis{[2-(diethylamino)ethyl]amino}anthracene-9,10-dione,1,4-bis{[2-(2-hydroxyethylamino)ethyl]amino}-anthracene-9,10-dione and1-(2-(diethylamino)ethyl]amino)-4-{[2-(2-hydroxyethylamino)ethyl]amino}anthracene-9,10-dionefrom which the last mentioned compound may be separated, for example bychromatography. On oxidation, only the tertiary nitrogen atom of the[2-(diethylamino)ethyl)] amino group will be converted to N-oxide form.

Where one or more substituents X is present it may be appropriate,depending on the route of synthesis, to have these present throughout intheir final form or to generate the desired groups at a later stage inthe synthesis. Ether and ester groups X may of course readily beprepared by modification of hydroxy groups according to knownprocedures, precursors containing a hydroxy group X more often beingdescribed in the literature than those containing a corresponding etheror ester substituent.

It will be appreciated, however, that various alternative methods forthe preparation of the compounds (I) and intermediates therefor may beused as will be apparent in particular from the literature relating tosuch intermediates. Further details of the preparation of intermediatesfor the preparation of the compounds (I) of the present invention are tobe found in U.S. Pat. No. 4,197,249 and U.K. Patent GB 2,004,293Breferred to hereinbefore.

Certain of the intermediates corresponding to compounds (I) describedherein but without the tertiary amine group(s) in N-oxide form are noveland are within the scope of this invention. Such intermediates includeparticularly those of formula (Ia) in which at least one of R₁ to R₄ isa group NH--A--NR'R" and at least one other is a different groupNH--A--NR'R" or a group NH--A--NHR.

The compounds (I) may be formulated with a physiologically acceptablediluent or carrier for use as pharmaceuticals for both veterinary, forexample in mammals, and particularly human use by a variety of methods.For instance, they may be applied as a composition incorporating aliquid diluent or carrier, for example an aqueous or oily solution,suspension or emulsion, which may often be employed in injectable formfor parenteral administration and therefore may conveniently be sterileand pyrogen free. Oral administration may also be used and althoughcompositions for this purpose may incorporate a liquid diluent orcarrier, it is more usual to use a solid, for example a conventionalsolid carrier material such as starch, lactose, dextrin or magnesiumstearate. Such solid compositions may take the form of powders but aremore conveniently of a formed type, for example as tablets, cachets, orcapsules (including spansules). Alternative, more specialized types offormulation include liposomes and nanoparticles.

Other types of administration than by injection or through the oralroute which are of use in both human and veterinary contexts include theuse of suppositories or pessaries. Another form of pharmaceuticalcomposition is one for buccal or nasal administration or alternativelydrops for administration into the eye which may conveniently contain asterile liquid diluent or carrier. Other formulations for topicaladministration include lotions, ointments, creams, gels and sprays.

Compositions may be formulated in unit dosage form, i.e. in the form ofdiscrete portions containing a unit dose or a multiple or sub-unit of aunit dose.

Whilst the dosage of the compound used will vary according to theactivity of the particular compound and the condition being treated, itmay be stated by way of guidance that a dosage selected in the rangefrom 0.1 to 20 mg/kg per body weight per day, particularly in the rangefrom 0.1 to 5 mg/kg of body weight per day, will often be suitablealthough higher doses than this, for example in the range from 0.1 to 50mg/kg of body weight per day (or possibly even as high as described inU.S. Pat. No. 4,197,249) may be considered in view of the lower level oftoxic side effects obtained with the compounds (I). This dosage regimemay be continued for however many days is appropriate to the patient inquestion, the daily dosages being divided into several separateadministrations if desired. Thus, for example, in the case of conditionssuch as advanced breast cancer, non-Hodgkin's lymphyoma and hepatoma,treatment for one day followed by a repeated dose after an interval,such as 21 days, may be appropriate whilst for the treatment of acutenon-lymphocytic leukaemia, treatment over 5 consecutive days may be moresuitable.

The compounds (I) are of particular value for the treatment of cancer inwarm blooded animals including humans. The compounds are of interest inrelation to the treatment of solid tumours, such as various forms ofsarcoma and carcinoma, and also for disseminated tumours such asleukaemias. Areas of particular interest are the treatment ofnon-Hodgkins lymphoma, of breast cancer, and of acute non-lymphocyticleukaemia. In the treatment of cancer parenteral and sometimes topicaladministration is often of particular interest. Moreover, it may beadvantageous to use the compounds (I) in a combined treatment, givenseparately or together in the same composition, with other anti-canceragents, such as mitotic inhibitors, for example vinblastine; alkylatingagents, for example cis-platin, carboplatin and cyclophosphamide; otherantimetabolites, for example 5-fluorouracil, cytosine arabinoside andhydroxyurea; intercalating antibiotics, for example adriamycin andbleomycin; enzymes, for example asparaginase; topoisomerase inhibitors,for example etoposide and biological response modifiers, for exampleinterferon.

In a variation of the usual procedure which relies upon the anaerobicconditions within neoplastic tissue to effect selective reduction of theN-oxide in such tissue, the selectivity as between neoplastic and normaltissue can be enhanced. Thus antibodies can be raised against tumours byconventional procedures, particularly using hybridoma technology, andlinked covalently to a reductase using one of various conventionallinking agents. The conjugate is administered to the patient when itlocalises in the body at the tumour site and the compound (I) is thenadministered, the action of the reductase enhancing the specificity ofthe action of the compound at the tumour site.

The present invention thus includes a method of aiding regression andpalliation of a cancer sensitive to treatment with a compound of formulawhich comprises administering to a patient a therapeutically effectiveamount of a compound (I) as defined hereinbefore.

In addition to their anti-cancer use the compounds (I) are of interestfor various other pharmaceutical applications in view of their activityas chelating agents.

The invention is illustrated by the following Examples.

EXAMPLES Example 1 Preparation of1,5-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione (1)1,5-bis-{[2-(diethylamino)ethyl]amino}anthracene-9,10-dione

A solution of 5.52 g (0.02 mol) of 1,5-dichloroanthracene-9,10-dione in23.2 g (0.2 mol) of 2-(diethylamino)ethylamine is heated at refluxtemperature for 4 hours. The mixture is cooled in an ice-bath and 100 mlconcentrated hydrochloric acid is added with stirring. This acidicmixture is extracted with three aliquots of 200 ml of diethyletherfollowed by three aliquots of 200 ml of chloroform. The aqueous layer iscollected and made alkaline with sodium hydroxide solution, extractedinto chloroform and evaporated in vacuo at 30° C. The oily residue iswashed with water, dissolved in methanol and titrated with hydrogenchloride in dry diethyl ether to give a precipitate which is dried toyield 5.2 g of the title compound as the hydrochloride in the form of adark red solid, m.p. 158.5°-159.5° C.; λ_(max) (deionised water)(E/cm/M) 231 nm (34280), 516 nm (10690).

(2) 1,5-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione

A solution of 1.25 g (0.004 mol) of1,5-{2-(diethylamino)-ethyl]amino}anthracene-9,10-dione free base(prepared by washing the oily residue of (1) with water and drying) in20 ml of chloroform is cooled in an ice bath while stirring. Thissolution is treated with 1.2 g of 3-chloroperbenzoic acid, allowed tocome to room temperature and left for 18 hours protected from light. Themixture is evaporated in vacuo to a small volume and is subject to flashchromatography using a column of 70-230 mesh (60 A) silica gel and aneluting solvent of chloroform:methanol (5:1 v/v) to yield 0.45 g of thetitle compound. m.p. (following recrystallisation from chloroform togive dark red crystals) 125°-129° C. (decomposition): λ_(max) (deionisedwater) (E/cm/M) 231 nm (44030), 516 nm (11330).

EXAMPLE 2 Preparation of1,4-bis-{[2-(diethylamino-N-oxide)ethyl]amino}-anthracene-9,10-dione

(1) 1,4-bis-{[2-(diethylamino)ethyl]amino}anthracene-9,10-dione

A mixture of 5 g (0.021 mol) of 1,4-dihydroxyanthracene-9,10-dione and 2g (0.014 mol) of sodium dithionite in 20 ml water is stirred whilstheating under nitrogen at 90° C. until the mixture turns from orange tobrown indicating the presence of2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione. To this reactionmixture is added dropwise 20 g (0.17 mol) of 2-(diethylamino)-ethylamineover a 30 minute period. The mixture is heated at 50°-55° C. for 2 hoursand 20 ml of ethanol are added. The solution is then aerated and 200 mlof 2M hydrochloric acid are added. The acidic mixture is washed with3×200 ml of diethylether followed by 3×200 ml of chloroform. The aqueousphase is made alkaline by the addition of sodium hydroxide solution andextracted with 3×200 ml chloroform. The mixture is evaporated in vacuoto a 5 ml volume and subjected to flash chromatography using a column of70-230 mesh (60 A) silica gel (SiO₂) and an eluting solvent ofchloroform followed by chloroform:methanol (1:1 v/v). The blue blackmaterial removed from the column in chloroform/methanol is evaporated invacuo to yield 0.7 g of the title compound, m.p. 107.5°-108.5° C.;λ_(max) λ_(max) (deionised water) (E/cm/M) 256 nm (50950), 584 nm(18650).

(2)1,4-bis-{2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-diethylamino)ethyl-aminolanthracene-9,10-dionein 15 ml of dry chloroform is added 1.5 g (0.008 mol) of3-chloroperbenzoic acid. The mixture is stirred for 30 minutes, allowedto come to rom temperature and the left for 18 hours protected fromlight. The mixture is evaporated in vacuo to a 5 ml volume and subjectedto reverse phase chromatography using as column of octadecylsilanebonded to silica gel, 10 μM particle size, and methanol:ammoniumformate, 0.5M, pH 4.25 (30:70 v/v), as an eluting solvent. The eluatefractions identified by thin-layer chromatography (chloroform/methanol1:1 v/v) to contain a single dark blue-coloured component are pooled,extracted with chloroform and evaporated in vacuo. Drying overphosphorus pentoxide yields 0.17 g of the title compound as a dark bluesolid, m.p. 115°-118° C. (decomposition): λ_(max) (deionised water)(E/cm/M) 256 nm (20900), 584 nm (5880). Example 3 Preparation of1-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione (1)1-{2-(diethylamino)ethyl]amino}anthracene-9,10-dione

A mixture of 12.1 g (0.05 mol) of 1-chloroanthraquinone and 58 g (0.5mol) of 2-(diethylamino)ethylamine is heated under reflux for 2 hours.The reaction product is worked up as described in Example 1 except thatthe oily residue is only washed with water and then dried to yield 3.24g of the title compound as the free base in the form of an orange-redsolid, m.p. 98° C.; λ_(max) (deionised water) (E/cm/M) 248 nm (27180),493 nm (5770).

(2) 1-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione

1.25 g (0.0039 mol) of1-{[2-(diethylamino)ethyl]amino}anthracene-9,10-dione and 1.2 g(0.0068M) 3-chloroperbenzoic acid are reacted for 18 hours as describedin Example 1. Silica gel flash chromatography as in Example 1 but withchloroform/methanol (1:5 v/v) as eluting solvent gives an eluate whichis evaporated in vacuo. Recrystallisation of the residue from chloroformyields 0.45 g of the title compound as an orange-red solid, m.p.120°-125° C. (decomposition); λ_(max) (deionised water) (E/cm/M) 248 nm(32330), 493 nm (6180).

Example 4 Preparation of1,8-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione (1)1,8-bis-{(2-(diethylamino)ethyl]amino}anthracene-9,10-dione

A mixture of 5.52 g (0.02 mol) of 1,8-dichloroanthraquinone and 23.2 g(0.2M) 2-(diethylamino)ethylamine is heated under reflux for 3 hours.The reaction product is washed up as described in Example 1 except thatthe oily residue is only washed with water and then dried to yield 3.63g of the title compound as the free base in the form of a purple solid,m.p. 103.5°-106.5° C.; λ_(max) (deionised water) (E/cm/M) 236 nm(54940), 542 nm (13200).

(2) 1,8-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione

1.25 g (0.003 mol) of1,8-{[2-(diethylamino)ethyl]amino}-anthracene-9,10-dione and 2.5 g(0.0135 mol) 3-chloroperbenzoic acid are reacted for 4 hours asdescribed in Example 1. Flash chromatography as in Example 1 but usingchloroform/methanol (1:1 v/v) eluting solvent gives an eluate which isevaporated in vacuo. Recrystallisation of the residue from chloroformyields 0.18 g of the title compound as a purple solid m.p. 118°-119° C.(decomposition): λ_(max) (deionised water) (E/cm/M) 236 nm (39530), 542nm (8360).

Example 5 Preparation of1,4-bis-{[2-(diethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione(1)1,4-bis-{[2-(diethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione

A mixture of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone and 5 g (0.043 mol) ofN,N-diethylaminoethylamine in methanol is heated for 10 minutes toreflux temperature. The ethanol and unreacted N,N-diethylaminoethylamineare removed by distillation in vacuo and the remaining solidrecrystallised from ethyl acetate to yield 0.64 g of the title compoundas a dark blue solid, m.p. 203° C. as the dihydrochloride salt; λ_(max)(distilled water) (E/cm/M) 239 nm (44910), 605 nm (10890), 659 nm(10710).

(2)1,4-bis-{[2-(diethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

0.108 g (0.00023 mol) of1,4-bis-{[2-(diethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dioneis dissolved in 5 ml dichloromethane and the solution is cooled in anice-bath whilst stirring. The solution is treated with 0.2 g (0.000115mol) of 3-chloroperbenzoic acid, allowed to come to room temperature andleft for 18 hours protected from light. The mixture is then subjected toflash column chromatography using a column of silica gel (60A) and astep-gradient eluting solvent of dichloromethane:methanol:triethylaminestarting with dichloromethane:triethylamine (99:1 v/v) followed bydichloromethane:methanol:triethylamine (90:9:1 v/v/v) thendichloromethane:methanol:triethylamine (49.5:49.5:1 v/v/v) and finallymethanol:triethylamine (99:1 v/v). The major eluting blue fraction iscollected, filtered and evaporated in vacuo to yield 0.08 g of the titlecompound as a dark blue solid, m.p. 155°-158° C. (decomposition);λ_(max) (distilled water) (E/cm/M) 240 nm (15690), 609 nm (8809), 662 nm(5750).

Example 6 Preparation of1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione(1)1,4-bis-{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A solution of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone in methanol is refluxed undernitrogen. To this is slowly added 2 g (0.023 mol) ofN,N-dimethylaminoethylamine and the mixture is stirred at 50° C. for 1hour. The mixture is then stirred for 16 hours in air. The ethanol andunreacted N,N-dimethylaminoethyl amine are removed by distillation invacuo and the remaining solid is recrystallised from ethylacetate/methanol (2:1 v/v) to yield 0.68 g of the title compound as adark blue solid, m.p. 199°-200.5° C. as the dihydrochloride salt,λ_(max) (distilled water) (E/cm/M) 242 nm (43270), 606 nm (18050), 658nm (16220).

(2)1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A solution of 0.10 g (0.000242 mol) of1,4-bis-{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dionein 5 ml dichloromethane is cooled in an ice-bath whilst stirring. Tothis solution is added 0.2 g (0.000115 mol) of 3-chloroperbenzoic acidand the product is allowed to come to room temperature and left for 8hours protected from light. The mixture is then subjected to flashcolumn chromatography using a column of silica gel (60A) and astep-gradient eluting solvent of dichloromethane:methanol:triethylaminestarting with dichloromethane:methanol (50:50 v/v) followed bydichloromethane:methanol (20:80 v/v) and finally methanol:triethylamine(99:1 v/v). The last eluting blue fraction is collected, filtered andevaporated in vacuo to yield 0.075 g of the title compound as a darkblue solid, m.p. 124°-128° C. (decomposition); λ_(max) (distilled water)(E/cm/M) 222 nm (15500), 612 nm (6110), 664 nm (3685).

Example 7 Preparation of1,4-bis-{[2-(diethylamino-N-oxide)propyl]amino}-5,8-dihydroxyanthracene-9,10-dione(1)1,4-bis-{[2-(diethylamino)propyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A mixture of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone in 20 ml of aqueous potassiumcarbonate (5% w/v) and 0.2 g of sodium dithionite is stirred and flushedwith nitrogen. The mixture is then treated with 4 g (0.003 mol) ofN,N-diethylaminopropylamine and stirred at 80° C. for 18 hours in air.The ethanol and unreacted N,N-diethylaminopropylamine are removed bydistillation in vacuo and the remaining solid is recrystallised fromethyl acetate to yield 0.8 g of the title compound as a dark blue solid.m.p. 126°-128° C.; λ_(max) (distilled water) (E/cm/M) 241 nm (15370),611 nm (12460), 668 nm (11280).

(2)1,4-bis-{[2-(diethylamino-N-oxide)propyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A solution of 0.10 g (0.000242 mol) of1,4-bis-{[2-(diethylamino)propyl]amino}-5,8-dihydroxyanthracene-9,10-dioneis dissolved in 5 ml dichloromethane and cooled in an ice-bath whilststirring. The solution is treated with 0.2 g (0.000115 mol) of3-chloroperbenzoic acid, allowed to come to room temperature and leftfor 8 hours protected from light. The mixture is then subjected to flashcolumn chromatography using a column of silica gel (60A) and astep-gradient eluting solvent of dichloromethane:methanol:triethylaminestarting with dichloromethane:methanol (50:50 v/v) followed bydichloromethane:methanol (20:80 v/v) and finally methanol:triethylamine(99:1 v/v). The last eluting blue fraction is collected, filtered andevaporated in vacuo to yield 0.8 g of the title compound as a dark bluesolid which remains as a solid in a dessicator but becomes sticky onattempting a melting point determination; λ_(max) (distilled water)(E/cm/M) 248 nm (14220), 614 nm (8600), 666 nm (5144).

Example 8 Preparation of1-{[2-(diethylamino-N-oxide)ethyl]amino}-4-{[2-[(2-hydroxyethyl)amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione(1)1-{[2-(diethylamino)ethyl]amino}-4-{[2-[(2-hydroxyethyl)-amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A solution of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone in 10 ml ethanol is heated at50° C. under nitrogen with 2.1 g (0.00184 mol) ofN,N-diethylaminoethylamine and 1.9 g (0.00184 mol) of2-hydroxyethylaminoethylamine for 6 hours and then stirred in air for 16hours. The ethanol and unreacted N,N-diethylaminoethylamine and2-hydroxyethylaminoethylamine are removed by distillation in vacuo. Theremaining solid is dissolved in dichloromethane:methanol:0.3% w/vaqueous ammonia (96:3:1 v/v/v) and subjected to flash chromatographyusing silica gel (60A) and dichloromethane:methanol:aqueous ammonia(96:3:1 v/v/v) as eluting solvent. The major eluting fraction isevaporated in vacuo and the solid again column chromatographed on silicagel (60A) using a step gradient of dichloromethane:methanol (50:50 v/v)followed by dichloromethane:methanol:aqueous ammonia (49.75:49.75:0.5v/v/v). The major eluting blue fraction is collected, filtered andevaporated in vacuo to yield 0.2 g of the title compound as a dark bluesolid, m.p. 165°-167° C.; λ_(max) (distilled water) (E/cm/M) 232 nm(3380), 608 nm (20782), 660 nm (18900).

(2)1-{[2-(diethylamino-N-oxide)ethyl]amino}-4-{[2-[(2-hydroxyethyl)amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A solution of 0.10 g (0.00023 mol) of1-{[2-(diethylamino)-ethyl]amino}-4-{[2-[(2-hydroxyethyl)amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dioneis dissolved in 5 ml dichloromethane and cooled in an ice-bath whilststirring. This solution is treated with 0.1 g (0.000058 mol) of3-chloroperbenzoic acid, allowed to come to room temperature and leftfor 18 hours protected from light. The mixture is then subjected toflash column chromatography using a column of silica gel (60A) and astep-gradient eluting solvent as described for Example 6. The lasteluting blue fraction is collected, filtered and evaporated in vacuo toyield 0.06 g of the title compound as a dark blue solid, m.p. 92°-93.5°C. (decomposition); λ_(max) (phosphate buffer pH 7.4) (E/cm/M) 238 nm(8203), 607 nm (6396), 658 nm (4733).

Example 9 Preparation of 1-{[2-(diethylamino-N-oxide)ethyl]amino}-4-{[2-(ethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione 1)1-{([2-(diethylamino)ethyl]amino}-4-{[2-(ethylamino)ethyl]-amino}-5,8-dihydroxyanthracene-9,10-dione

A suspension of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone in 10 ml of propan-2-ol isstirred at 50° C. under nitrogen. To the mixture is added 2.1 g (0.00184mol) of N,N-diethylaminoethylamine and 1.6 g (0.00184 mol) ofN-ethylaminoethylamine the product is stirred in for 12 hours undernitrogen and then for a further 6 hours in air. The ethanol andunreacted N,N-diethylaminoethylamine and N-ethylaminoethylamine areremoved by distillation in vacuo and the remaining solid isrecrystallised from ethyl acetate to yield 0.2 g of the title compoundas a dark blue solid m.p. 161°-163° C.; λ_(max) (distilled water)(E/cm/M) 242 nm (15520), 609 nm (10196), 659 nm (12640).

(2)1-{[2-(diethylamino-N-oxide)ethyl]amino)-4-{[2-(ethylamino)-ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

0.10 g (0.00023 mol) of1-{[2-(diethylamino)ethyl]amino}-4-{[2-(ethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dioneis dissolved in 5 ml dichloromethane and the solution is cooled in anice-bath whilst stirring. To the solution is added 0.1 g (0.000058 mol)of 3-chloroperbenzoic acid and it is then allowed to come to roomtemperature and left for 16 hours protected from light. The mixture isthen subjected to flash column chromatography using a column of silicagel (60A) and a step-gradient eluting solvent ofdichloromethane:methanol (90:10 v/v) followed bydichloromethane:methanol:0.3% w/v aqueous ammonia (90:9:1 v/v/v). Thelast eluting blue fraction is collected, filtered and evaporated invacuo to yield 0.08 g of the title compound as a dark blue solid, whichremains as a solid in a dessicator but becomes sticky on attempting amelting point determination; λ_(max) (distilled water) (E/cm/M) 244nm(14776), 612 nm (10080), 662 nm (6532).

Example 10 Preparation of 1,4-bis-{[2-(di(2-hydroxyethyl)amino)-N-oxideethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (1)1,4-bis-{[2-(di(2-hydroxyethyl)amino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A mixture of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone and 4 g (0.003 mol) ofN,N-di(2-hydroxyethyl)aminoethylamine treated as described in Example 7produces a dark solid. This is dissolved in 10 ml ofmethanol:dichloromethane (50:50 v/v) subjected to silica gel (60A) flashchromatography using a step gradient of dichloromethane:methanol (90:10v/v) followed by dichloromethane:methanol (50:50 v/v). The major elutingfraction is collected, filtered and evaporated in vacuo to yield thetitle compound as a dark blue solid, which remains as a solid in adessicator but becomes sticky on attempting a melting pointdetermination; λ_(max) (distilled water) (E/cm/M) 235 nm (12726), 605 nm(7236), 655 nm (6396).

(2)1,4-bis-{[2-(di(2-hydroxyethyl)amino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

0.09 g (0.00017 mol) of1,4-bis-{[2-(di(2-hydroxyethyl)ethyl]-amino}-5,8-dihydroxyanthracene-9,10-dioneis dissolved in 5 ml dichloromethane and the solution is cooled in anice-bath whilst stirring. To the solution is added 0.1 g (0.00044 mol)of 3-chloroperbenzoic acid and it is then allowed to come to roomtemperature and left for 16 hours protected from light. The reactionmixture is filtered and the solid washed five times with 25 ml aliquotsof dichloromethane followed five times by 10 ml aliquots of methanol.The residue is filtered to yield 0.03 g of the title compound as a darkblue solid, m.p. 134.5°-135.5° C.; λ_(max) (distilled water) (E/cm/M)238 nm (24527), 610 nm (9331), 666 nm (5435).

Example 11 Preparation of1-{[2-(dimethylamino-N-oxide)ethyl]amino}-4-{[2-[2-hydroxyethyl)amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione(1)1-{[2-(dimethylamino)ethyl]amino)-4-{[2-[2-hydroxyethyl)amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione

A mixture of 1.0 g (0.0037 mol) ofleuco-1,4,5,8-tetrahydroxyanthraquinone, 1.6 g (0.00184 mol) ofN,N-dimethylaminoethylamine and 1.9 g(0.00184 mol) of2-hydroxyethylaminoethylamine in ethanol is stirred under nitrogen atroom temperature for 3 hours. The mixture is then stirred for 16 hoursin air. The ethanol and unreacted N,N-dimethylaminoethylamine and2-hydroxyethylaminoethylamine are removed by distillation in vacuo. Theresulting solid is dissolved in dichloromethane:methanol:0.3% w/vaqueous ammonia (49.75:49.75:0.5 v/v/v) and subjected to columnchromatography on silica gel (60A). The chromatography procedure isrepeated using dichloromethane:methanol:triethylamine (90:9:1 v/v/v) andthe major eluting fraction is collected, filtered and evaporated invacuo to yield 0.260 g of the title compound as a dark blue solid, m.p.136°-140° C. as the dihydrochloride; λ_(max) (distilled water) (E/cm/M)244nm (30430),607nm (15683), 658nm (13560).

(2)1-{[2-(dimethylamino-N-oxide)ethyl]amino}-4-{[2-[2-hydroxyethyl)amino]ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione

0.10 g (0.00023 mol) of1-{[2-(dimethylamino)ethyl]amino}-4-{[2-[(2-hydroxyethyl)amino]ethyl]amino}-5,8-dihydroxyanthracene-9,10-dioneis dissolved in 5 ml of dichloromethane and the solution is cooled in anice-bath whilst stirring. To this solution is added 0.1 g (0.000058 mol)of 3-chloroperbenzoic acid and it is then allowed to come to roomtemperature and left for 18 hours protected from light. The mixture isthen treated as described in Example 8 to yield 0.03 g of the titlecompound as a dark blue solid, m.p. 128°-132° C. (decomposition);λ_(max) (phosphate buffer pH 7.4) (E/cm/M) 240 nm(13243), 610 nm(6475),664 nm(5915).

Example 12: Biological Activity

The cytotoxicity was compared of the four compounds

(a) 1,8-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione

(b) 1,4-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione,

(c) 1-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione, and

(d) 1,5-bis-{[2-(diethylamino-N-oxide)ethyl]amino}anthracene-9,10-dione,

under aerobic and anaerobic conditions and a comparison was also madewith the cytotoxicity of the corresponding compound in which theterminal, tertiary nitrogen atom is not in N-oxide form.

Cells of the MCF-7 human breast cancer cell line (5×10⁵ /ml) were seededinto 12-well culture plates containing RPMI 1640 medium (Flow Labs.,Irving, Scotland) supplemented with 10% v/v foetal bovine serum andgrown at 37° C. to confluence. In the aerobic experiments, the cellswere treated with one of a range of concentrations of the compound andthen incubated in air for 24 hours. In the anaerobic experiments,following treatment of the cells with compound the culture plates wereplaced in a gas-tight chamber (Flow Labs) which was flushed withnitrogen 30 minutes, the cells then being incubated for 24 hours undernitrogen. Following both types of experiment the cells were washed freeof the compound with isotonic saline and were then grown for a further 3days in air. The surviving monolayer cells were counted using a Coultercounter.

The results obtained are presented in the Figure, the different parts(a) to (d) of which correspond to the four compounds identified above bythese letters. In each part of the Figure plot 1 corresponds to theN-oxide under aerobic conditions, plot 2 corresponds to the N-oxideunder anaerobic conditions and plot 3 corresponds to the correspondingtertiary amine under anaerobic conditions.

The Figure shows that each of the N-oxides is substantiallynon-cytotoxic in air but these compounds are cytotoxic when cells areexposed to them in nitrogen, although the level of cell kill observed isnot necessarily fully equivalent to the level resulting from the use ofan equimolar amount of the parent tertiary amines under the sameconditions.

I claim:
 1. A compound of formula (I) ##STR7## in which R₁, R₂, R₃ andR₄ are each separately selected from the group consisting of hydrogen,X, NH--A--NHR and NH--A--N(O)R'R" wherein X is hydroxy, halogeno, amino,C₁₋₄ alkoxy or C₂₋₈ alkanoyloxy, A is a C₂ -4 alkylene group with achain length between NH and NHR or N(O)R'R" of at least 2 carbon atomsand R, R' and R" are each separately selected from the group consistingof C₁₋₄ alkyl groups and C₂₋₄ hydroxyalkyl and C₂₋₄ dihydroxyalkylgroups in which the carbon atom attached to the nitrogen atom does notcarry a hydroxy group and no carbon atom is substituted by two hydroxygroups, or R' and R" together are a C₂₋₆ alkylene group which with thenitrogen atom to which R' and R" are attached forms a heterocyclic grouphaving 3 to 7 atoms in the ring, but with the proviso that at least oneof R₁ to R₄ is a group NH--A--N(O)R'R", the compound optionally being inthe form of a physiologically acceptable salt.
 2. A compound accordingto claim 1, in which R₁, R₂, R₃ and R₄ are each separately selected fromthe group consisting of hydrogen, hydroxy, NH--A--NHR andNH--A--N(O)R'R".
 3. A compound according to claim 1 or 2, in which A isethylene.
 4. A compound according to claim 1, 2 or 3, in which R, R' andR" are each separately selected from the group consisting of CH₃, CH₂CH₃, CH₂ CH₂ CH₃, CH₂ CH₂ OH, CH₂ CH₂ CH₂ OH, CH(CH₃)CH₂ OH and CH₂CHOHCH₂ OH.
 5. A compound according to claim 1, which contains one ortwo groups NH--A--N(O)R'R", these being separately selected fromNH--(CH₂)₂ --N(O)(CH₃)₂, NH--(CH₂)₂ -N(O)(CH₃)C₂ H₅, NH--(CH₂)₂--N(O)(C₂ H₅)₂, NH--(CH₂)₂ --N(O)(CH₂ CH₂ OH)₂, NH--(CH₂)₂ --N(O)(CH₂CH₂ CH₂ OH)₂, NH--(CH₂)₂ --N(O)CH(CH₃)OH and NH--(CH₂)₂ --N(O)(CH₂CHOHCH₂ OH)₂.
 6. A compound according to claim 1, which contains onegroup NH--A--N(O)R'R" and one group NH--A--NHR, this latter group beingselected from the group consisting of NH--(CH₂)₂ --NHCH₃, NH--(CH₂)₂--NHC₂ H₅, NH--(CH₂)₂ --NHCH₂ CH₂ OH, NH--(CH₂)₂ --NHCH₂ CH₂ CH₂ OH,NH--(CH₂)₂ --NHCH(CH₃)CH₂ OH and NH--(CH₂)₂ --NHCH₂ CHOHCH₂ OH.
 7. Acompound according claim 1, in which(1) R₁ =NH--A--N(O)R'R" at position1, R₂ =H, R₃ =R₄ =OH at positions 5 and 8; (2) R₁ =NH--A--N(O)R'R" atposition 1, R₂ =OH at position 4, R₃ =OH at position 5 or position 8 andR₄ =H; (3) R₁ =NH--A--N(O)R'R" at position 1 and R₂ =R₃ =R₄ =OH atpositions 4, 5 and 8; (4) R₁ =R₃ =NH--A--N(O)R'R" at positions 1 and 8and R₂ =R₄ =OH at positions 4 and 5; (5) R₁ =R₂ =NH--A--N(O)R'R" atpositions 1 and 4 and R₃ =R₄ =OH at positions 5 and 8; or (6) R₁ =R₃=NH--A--N(O)R'R" at positions 1 and 5 and R₂ =R₄ =OH at positions 4 and8.
 8. A compound according to claim 6, in which(1) R₁ =NH--A--N(O)R'R"at position 1, R₂ =NH--A--NHR at position 4, and R₃ =R₄ =OH at positions5 and 8; (2) R₁ =NH--A--N(O)R'R" at position 1, R₂ =OH at position 4, R₃=NH--A--NHR at position 5 and R₄ =OH at position 8, or (3) R₁=NH--A--N(O)R'R" at position 1, R₂ =R₃ =OH at positions 4 and 5 and R₄=NH--A--NHR at position
 8. 9. A compound according to claim 1, in whichR₁ =R₂ =NH--A--N(O)R'R" at positions 1 and 4 and R₃ =R₄ =OH at positions5 and 8, or R₁ =R₃ =NH--A--N(O)R'R" at positions 1 and 5 and R₂ =R₄ =OHat positions 4 and 8 with both NH--A--N(O)R'R" being NH--(CH₂)₂N(O)(CH₃)₂ or NH--(CH₂)₂ N(O)(CH₂ CH₂ O)₂.
 10. A compound according toclaim 1, in which R₁ =NH--A--N(O)R'R" at position 1, R₂ --NH--A--NHR atposition 4 and R₃ =R₄ =OH at positions 5 and 8 or R₁ =NH--A--N(O)R'R" atposition 1, R₂ =OH at position 4, R₃ =NH--A--NHR at position 5 and R₄=OH at position 8 with NH--A--N(O)R'R" being NH--(CH₂)₂ N(O)(CH₃)₂ orNH--(CH₂)₂ N(O)(CH₂ CH₂ OH)₂ and NH--A--NHR being NH--(CH₂)₂ NHCH₃ orNH(CH₂)₂ NHCH₂ CH₂ OH.
 11. A pharmaceutical composition comprising acompound of formula (I) as defined in claim 1 together with aphysiologically acceptable diluent or carrier.
 12. A method of aidingregression and palliation of a cancer which comprises administering to apatient a therapeutically effective amount of a compound of formula (I)as defined in claim
 1. 13. A compound according to claim 1, which is1,4-bis-{[2-(diethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione.